! SUMMARY The RAG1/RAG2 recombinase, which initiates V(D)J recombination, is a defining feature of jawed vertebrate adaptive immunity and is thought to have evolved from a transposable element. Key aspects of RAG biochemistry and in vivo regulation are not understood, leaving large gaps in our understanding of the mechanisms by which RAG contributes to genome instability and the development of cancer. The Transib and ProtoRAG transposons, which encode RAG-like transposases, provide an entirely new toolbox with which to fill these gaps. In unpublished work, we have: i) determined the structure of ProtoRAG-DNA complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that diffract x-rays to ~3 resolution; iii) identified a key component of the mechanism that directs coordinated (coupled) DNA cleavage by RAG; and iv) discovered two mechanisms that suppress RAG-mediated transposition in vivo. We will use these novel tools and findings to accomplish our central objective: to determine the biochemical, structural, and regulatory mechanisms that have evolved to orchestrate RAG function and to ascertain the biological consequences of failures of these mechanisms. Our proposal is organized around three core questions. First, what mechanisms explain coupled cleavage by RAG and why do those mechanisms break down? Second, how do the different modules within RAG work together to determine activity? And third, what protects the genome from RAG-mediated transposition and what are the consequences when those mechanisms fail? These questions are addressed in two interwoven aims: Aim 1: Determine the underpinnings of DNA recognition and coupled cleavage by RAG and RAG- family transposases. ProtoRAG transposase binds and cleaves DNA in a manner with striking similarities to improperly regulated cleavage by RAG. Using novel RAG-ProtoRAG chimeric proteins, biochemistry, single molecule biophysics, and cryo-EM and x-ray crystallography, we will determine how DNA binding domains, DNA bending, complex stability, and conformational changes contribute to coordinated vs. uncoordinated cleavage in synaptic complexes formed by RAG and RAG-like transposases. Aim 2: Determine the regulation, targeting, and biological consequences of transposition into the mammalian genome by RAG. Building on our discovery of RAG mutants that uncouple DNA cleavage or activate transposition in vivo, we will use a suite of in vitro and in vivo transposition, cleavage, and high- throughput sequencing assays in normal and DNA repair-deficient cells to quantitate and map transposition mediated by intact and mutant RAG enzymes. In addition, we will generate and analyze RAG-mutant mice with regulatory defects in DNA cleavage and transposition. Together, our results will reveal how DNA repair factors and RAG catalytic and regulatory modules have evolved to protect genome stability and shield developing lymphocytes from malignant transformation during the process of V(D)J recombination.